光 - 電子相関顕微鏡法とアレイトモグラフィー法を組み合わせて蛍光を放つオルガネラを電顕で捉える

Abstract

Correlative light and electron microscopy (CLEM) is an analytical technique used to evaluate the correlation between fluorescence images of identical samples and regions of fluorescent-labeled organelles captured using light microscopy and electron microscopy. By utilizing various methods during sample preparation, the fluorescence images of fluorescent-labeled organelles are juxtaposed with ultra-structures of the same region obtained using scanning electron microscopy (SEM) or transmission electron microscopy. Unlike animal tissues and adherent cells, visualizing the organelles detected in plant samples via CLEM analysis is difficult because of the non-adherence of plant tissue and cultured cells to glass plates and the size of the cells. Therefore, we combine the serial section SEM method (Array tomography) based on ultra-thin sections and CLEM to identify fluorescent-labeled organelles in plant cells and reconstruct them in three dimensions (3D). In particular, as osmium (Os) epoxy-tolerant fluorescent proteins have recently been reported to be resistant to Os fixation and epoxy resin embedding in animal adherent cells, we applied these fluorescent tags to plant samples. In this paper, we introduce the sample preparation method for the CLEM analysis of organelles labeled with Os epoxy-tolerant fluorescent proteins in plant tissues, alongside the method for obtaining CLEM images and the combined 3D-CLEM analysis method comprising CLEM and array tomography.